1. Field of the Invention
The invention generally relates to a method for nucleic acid hybridization, and specifically relates to a method and apparatus with temperature control for nucleic acid hybridization and hydrogen bond denaturation to accelerate nucleic acid hybridization.
2. Related Art
Based on rapidly developed techniques, such as PCR (polymerase chain reaction) and nucleic acid hybridization, molecular biotechnology has been gradually integrated with different areas, including material science, bioinformatics, and electronic technology, to bring about a new area of Biochips. The emergence of biochips could significantly decrease needed detection time for some diseases. For example, the initial detection time of 3-5 days by cell culture techniques can be decreased to be less than 6 hours by utilizing biochip technology.
However, such 6-hour detection time still cannot satisfy the demands of certain diseases. For instance, some diseases take less than 2 days from diagnosis to cause death. Therefore, current biochip research has been focused on shortening the needed detection time of the biochip.
To shorten the detection time, it is necessary to start from the most time-consuming technique during the test. PCR takes approximately 1.5 hours and nucleic acid hybridization takes about 4 hours. Together, these two techniques account for 92% of the whole biochip detection time. Therefore, how to shorten the amount of time consumed by these two techniques has become a key point. Presently, numerous researchers have put forth much effort on this issue, and the invention is focused on the most time-consuming nucleic acid hybridization technique.
The underlying mechanism for techniques such as Hybridization Helper is to utilize an oligonucleoride (i.e. Helper), which is complementary to the upstream or downstream region of the area where probing nucleic acid is hybridized to sample nucleic acid. Such hybridization between the nucleic acid Helper and sample nucleic acid is employed to stretch the sample nucleic acid (to eliminate the original cluttered circular conformation that is unfavorable for hybridization), which help the hybridization reaction.
In addition, another technique (called nucleic acid precipitating reagents) is available. The underlying mechanism for this technique is to utilize various buffered salt solutions to promote the sample nucleic acid precipitation at the nearby area of the probing nucleic acid. Such methodology increases the sample nucleic acid concentration in the local area to promote the processing of the hybridization reaction.
There is still another available technique called the branched oligonucleoride multimer technique, which uses probes immobilized on the surface of a chip for catching sample nucleic acid. Then, a branched oligonucleoride multimer, which is complementary to the sample nucleic acid, is linked to sample nucleic acid. Finally, fluor- or radio-labeled nucleic acid detectors are complementarily hybridized with the branched oligonucleoride multimer. Since tens or hundreds of detectors can hybridize onto a branched oligonucleoride multimer, the detection intensity can be greatly increased and the hybridization time can therefore be shortened.
Furthermore, a technique called electrically controlled hybridization takes advantage of the characteristics of negatively charged nucleic acid. By immobilizing the positive pole near the nucleic acid probe, nucleic acid in samples is lured near to the probe to make the sample nucleic acid highly concentrated in a small area and accomplish the goal of accelerating hybridization.
Another technique called volume exclusion agents employs organic molecules to form a reticular macro-structure, which can expel part of the hybridization buffer to increase the local concentration of sample nucleic acid and therefore promote the hybridization reaction.
Similar to the above technique, one technique called amphipathic hydrocarbon polymer (AHP) utilizes bipolar organic molecules (hydrophilic and hydrophobic) to form a reticular macro-structure. This macro-structure can expel part of the hybridization buffer to increase the local concentration of sample nucleic acid and therefore promote the hybridization reaction.
An apparatus called the highly parallel-integrated microfluidic biochannel array has integrated various functions, including sample pre-treatment, PCR, hybridization, washing, and signal detection. However, the hybridization rate of this apparatus has not yet been improved.
Finally, in the apparatus called the dynamic hybridization system, a nucleic acid probe is fixed on a semipermeable membrane. Meanwhile, fluid (containing sample nucleic acid) is driven by air or vacuum compression to flow towards the semipermeable membrane. Sample nucleic acids are delayed when the fluid passes through the semipermeable membrane and sample nucleic acids accumulate around the membrane to yield a higher concentration of sample nucleic acid. The hybridization rate is increased because non-complementary nucleic acid is able to pass through the holes of the semipermeable membrane.
Most of the above mentioned methods increase the hybridization rate by increasing sample nucleic acid concentration, linearlizing samples, or employing branched structure. All these hybridization accelerating methods have limitations, such as only being operable on a large scale. Nevertheless, how to speed up nucleic acid hybridization while also making the process applicable on a small scale is still the focus of a great deal of effort in research.